Fluorescence Polarization as a Detection/Diagnostic Tool
Immunization strategies to specific disease causing organisms and toxins are being developed and implemented aimed at prophylactically inhibiting disease processes. However, many important public health related organisms remain for which no prophylactic measures are available or can be practicably administered on a wide scale. In these cases prophylactic screening is required to ensure early treatment.
Currently available methods for the detection of pathogen exposure, infection and diagnosis varies depending on the target organism. Most diagnostic methods, however, require a biological sample, such as blood or serum, to be obtained and tested for the presence of antibody specific to the target organism or for antigen. The assay methods generally performed are modifications to surface-binding, heterogeneous assays such as enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), or agglutination. Since these assays require the interaction of antigen with a specific surface, there is often exhibited significant high non-specific binding and concomitant loss of specificity.
Fluorescence polarization (FP) overcomes many of the inherent problems encountered with most surface-binding antibody-based assays. FP is the process in which visible or ultraviolet light is polarized with a filter and illuminates a fluorochrome attached region of a target molecule, that in turn fluoresces, emitting light of longer wavelength whose signal is captured and recorded (1). The emitted light retains its polarization in solutions containing slower turning, large molecule-fluorochrome complexes compared with the presence of smaller labeled molecules. Different fluorochromes can be chosen to accommodate molecules of different sizes up to 107 kDa molecular weight (2).
FP assays have been utilized to measure different types of binding reactions to follow proteolytic reactions with and without their inhibitors and to measure various other enzymatic or receptor binding reactions (3, 4). In clinical settings, FP is used to measure the level of drugs, hormones or antibodies in blood plasma (5). Furthermore, the value of using FP diagnostic methods for the diagnosis of infection using other, non-serum matrixes, such as oral fluids and saliva, is gaining recognition (6).
Consequential to the recognized uses of FP, methods utilizing FP for detection of a number of target moieties have been developed. U.S. patent to Wang, et al. (7) discloses a method and reagents for determining a ligand, particularly steroid, hormone, anti-asthmatic, anti-neoplastic, anti-arrhythmic, anti-convulsant, antibiotic, anti-arthritic, antidepressant, cardiac glycoside, or a metabolite thereof, in biological fluids such as serum, plasma, spinal fluid, amnionic fluid, and urine. In particular, Wang, et al. relates to a specific class of tracer compounds required as reagents in such procedures. Additionally, a U.S. patent to Jolley, et al. (8) discloses a homogeneous immunoassay in which a fluorophore-conjugated lipopolysaccharide derived bacterial antigen is reacted with antibodies specific for the antigens in a diluted serum specimen, with quantitative detection of the formation of an immune complex obtained by measuring the change in fluorescence polarization after complex formation.
U.S. patent to Nakayama, et al. (9) discloses a fluorescence polarization method for analyzing a target moiety in a given sample. The procedure employs the steps of: (a) providing a fluorescent-labeled protein in which a protein is covalently bound to a fluorochrome(s), wherein the protein is capable of specifically binding to the assay-object; (b) allowing the fluorescent-labeled protein to bind to the assay-object; and (c) measuring a change in the degree of fluorescence polarization which has taken place in the fluorescent-labeled protein by its binding to the assay-object.
Knowledge of the epidemiology of diseases is important in health care planning and treatment. Both planning and treatment are dependent upon accurate and rapid diagnosis. FP technology is highly amenable to the accurate estimation of concentrations of diagnostic markers, drugs and chemicals, or bio-hazardous agents, in clinical as well as environmental samples, using a range of different fluid matrixes.
Although analysis of serum for the presence of specific hormones, drugs, antibodies and antigens routinely used, saliva and oral fluids are biochemically distinct and have been increasingly recognized as acceptable alternatives. Unlike serum samples, oral fluids are collected without pain, needle sticks, or religious and social prohibitions, and their use involves minimal risk or exempt protocols for the use of human subjects. Therefore, the use of FP using serum or oral fluids can be utilized to accurately detect or diagnosis markers, drugs, chemicals or specific biohazardous agents within a few seconds to several minutes. Additionally, FP is useful for the evaluation of environmental samples including water sources for the presence of contaminating chemicals or microorganisms. The applicability of FP assay methods to the analysis of a broad spectrum of sample sources, unlike many antibody-based or molecular-based methods, is partially due to the relative robustness of FP which is unaffected by constituents in non-homogeneous samples, such as whole blood, saliva or even environmental samples. Specificity of FP assays can be readily designed to be generally very high, with a specificity approaching or exceeding approximately 98%.
A significant limitation of current FP methods, however, are the size and power requirements of available instrumentation. These constraints are particularly acute in field settings, small clinical settings or in third world health care environments where infrastructure is limited. Therefore, there has been a long-felt need for more compact and simplified FP devices. The advent of new, compact devices will permit an expansion of effective, noninvasive as well as accurate laboratory quality diagnostic tests performed in field settings.